Identification of the C5a des Arg cochemotaxin.
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Amino Acids↗
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Results for “Complement C5a, des-Arginine”
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Complement activation and the toxicity of stroma-free hemoglobin solutions in primates.
The toxicity of hemoglobin solutions was studied in the context of their ability to activate serum complement (C). Three bovine polymerized hemoglobin solutions (BPHSs) with different degrees of purity were used for experiments in vitro and in vivo. BPHS-1 contained bacterial endotoxins (E) (5 EU/ml) and stromal phospholipids (PLs) (1.2 mg/dl), BPHS-2 contained only PLs (2.0 mg/dl), while BPHS-3 was completely free of both contaminants. C-activation was studied by the direct measurement of C3a, C4a, and C5a des Arg fragments, using commercially available RIA kits. During 1 hour of incubation with fresh monkey plasma, BPHS-1 and -2 activated both pathways of C, while BPHS-3 caused no activation of any factor. In vivo, Hb solutions were used to replace one-third of blood volume in three groups of six Coebus monkeys each, while fresh homologous plasma was used in a control group of four animals. Impure solutions activated the alternative pathway of C and caused significant reactions of the circulating blood (thrombocytopenia, leukopenia, and disseminated intravascular coagulation) associated with multiorgan dysfunction (cardiac arrhythmias, hypoxemia, reduction of renal clearance of endogenous creatinine, and elevation of liver enzyme SGPT). The pure solution neither activated C nor caused any reaction in the circulating blood. However, it caused a moderate degree of direct tissue injury, evidenced by transient reduction of creatinine clearance and elevation of SGPT. These observations suggest that impure and pure Hb solutions carry separate mechanisms of toxicity. Complement, activated by toxic impurities, plays an active role in the toxicity of impure solutions. C-activation in vitro could be used as a screening test of biocompatibility.
Animals↗
[Immunologic defense mechanism of complement-derived inflammatory chemical mediators].
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Animals↗
A direct in vivo comparison of the inflammatory properties of human C5a and C5a des Arg in human skin.
C5a is an 11,000-Da complement-derived inflammatory glycoprotein that has been shown to mediate inflammatory reactions in vitro as well as in vivo in human skin. The C5a degradation product, C5a des Arg, is rapidly formed after exposure of C5a to serum carboxypeptidase N and may represent the relevant C5-derived inflammatory peptide in vivo. To examine the biologic activity of human C5a des Arg in vivo and to compare it with that seen with human C5a, we purified and characterized homogeneous preparations of human C5a and C5a des Arg and injected them intradermally into seven normal volunteers. C5a des Arg exhibited biochemical and biologic properties in vitro that were different from those of C5a. When injected into human skin, C5a des Arg was less potent than C5a, in respect to both minimal dose eliciting wheal and flare reactions and maximal wheal and flare elicited at a given dose, but C5a des Arg still elicited cutaneous wheal and flare reactions at physiologically relevant concentrations. Histologically, C5a des Arg skin test sites showed dense polymorphonuclear neutrophil-rich infiltrates associated with leukocytoclasis, dermal mast cell degranulation, and endothelial cell swelling. These were virtually indistinguishable from reactions elicited by C5a and occurred with concentrations attainable in vivo. Cutaneous wheal and flare reactions elicited by either C5a or C5a des Arg were partially inhibited by H1 antihistamines but were unaffected by selected nonsteroidal anti-inflammatory agents.
Aspirin↗
C3a(C3adesArg) induces production and release of interleukin 1 by cultured human monocytes.
Purified human C3a(C3adesArg) induced dose-dependent generation of intracellular IL 1 activity and release of IL 1 in cultures of human mononuclear adherent cells in serum-free conditions. Concentrations of C3a(C3adesArg) of 10(-8) M and 6 hr of culture were sufficient to induce production of cell-associated IL 1, as detected in monocyte lysates. Ten- to 100-fold higher concentrations of C3a(C3adesArg) and 24 hr of culture were required for induction of IL 1 release. Release of IL 1 induced by suboptimal amounts of C3a(C3adesArg) was greatly enhanced by the addition of indomethacin to the culture medium. Contamination with C5a of the C3a(C3adesArg) preparation did not account for C3a(C3adesArg)-induced IL 1 production. Induction of IL 1 activity by C3a(C3adesArg) was not due to contaminating LPS, as indicated by the following observations: the amount of contaminating LPS in C3a(C3adesArg) was below that which could induce IL 1 release from human monocytes in serum-free conditions; induction of IL 1 by C3a(C3adesArg) was not suppressed by polymyxin B; kinetics of IL 1 production and release in the presence of C3a(C3adesArg) differed from those observed in the presence of LPS; and sialated gangliosides, which inhibit IL 1 release induced by LPS, had no effect on the induction of IL 1 by C3a(C3adesArg). The C3a(C3adesArg) preparation used in this study mostly contained the desArg derivative, suggesting that, in contrast with the requirement for an intact C-terminal arginyl residue for the spasmogenic activity of C3a, both C3a and its C3adesArg derivative may interact with receptors on human monocytes. By inducing IL 1 production and release, C3a(C3adesArg) may contribute to the generation of the inflammatory process and the regulation of the immune response.
Cells, Cultured↗
Chemotactic peptides modulate adherence of human polymorphonuclear leukocytes to monolayers of cultured endothelial cells.
We have used a new centrifugation assay to examine the effects of highly purified human C5a and C5a des Arg, as well as effects of N-formyl-methionyl-leucyl-phenylalanine (FMLP), on both the extent and strength of human polymorphonuclear leukocyte (PMN) adherence to monolayers of cultured human umbilical vein endothelial cells. At concentrations that were chemotactic for PMN, C5a (0.1 nM), C5a des Arg (5.0 nM), and FMLP (1.0 nM) significantly reduced the percentage of PMN that adhered to endothelial monolayers. Adherence also was reduced by C5a des Arg that was generated by incubating (37 degrees C, 30 min) fresh human serum with either zymosan or purified C5a. High concentrations of C5a (greater than 1.0 nM) and FMLP (greater than 50 nM) that diminished PMN chemotaxis significantly enhanced the percentage of PMN that adhered tightly to endothelial cells (adherent cells resisted a dislodgment force of 1200 X G). Tight adherence of PMN to endothelial cells also was increased by high concentrations of C5a that were added to human serum in which carboxypeptidase N activity was destroyed by heating (56 degrees C, 30 min), and by C5a that was generated by incubating (37 degrees C, 30 min) fresh human serum with zymosan in the presence of the carboxypeptidase N inhibitor, epsilon-aminocaproic acid. High concentrations of C5a des Arg (up to 80 nM) neither enhanced adherence of PMN to endothelial cells nor decreased PMN migration. Thus, a reciprocal relation exists between PMN migration and PMN adherence to endothelial cells in response to chemotactic factors. At concentrations that are chemotactic for human PMN, C5-derived peptides and FMLP reduce the adherence of PMN to endothelial monolayers. Only at concentrations that decrease PMN migration do C5a and FMLP augment PMN adherence.
Adult↗
Polymorphonuclear leukocyte-dependent plasma leakage in the rabbit skin is enhanced or inhibited by prostacyclin, depending on the route of administration.
Experiments were designed to test for possible differing modulatory effects of either intravascular or extravascular prostaglandins (PGs) on local edema induced by intradermally injected inflammatory mediators in the rabbit. Local extravascular PGI2, PGE2 and 15-methyl-PGE1, with similar potencies, had a marked potentiating effect on local edema induced by C5a des Arg. Local extravascular PGI2 also potentiated edema when tested with leukotriene B4 (LTB4), bradykinin, and histamine. However, intravenously infused PGI2 at 50 ng/kg/min reversed the enhancing effect of local extravascular PGI2. At this dose the attenuating effect of PGI2 was selective for the polymorphonuclear (PMN) leukocyte-dependent edema induced by C5a des Arg and LTB4, but had no effect on edema induced by the nonchemoattractants histamine and bradykinin. Similarly, edema induced by N-formyl-methionyl-leucyl-phenylalanine was suppressed, but not that induced by platelet activating factor. 15-Methyl-PGE1, at 300 ng/kg administered systemically (subcutaneously), also selectively suppressed PMN-dependent edema. However, at higher doses of 3 and 60 micrograms/kg attenuation was nonselective and associated with a fall in systemic arterial blood pressure. These experiments demonstrate that the site of PG generation and action is an important determinant of its influence on edema formation.
Alprostadil↗
The phenomenon of leukergy: induction and detection of leukocyte aggregation in whole human blood.
Leukocyte aggregation is involved in the generation of vascular damage during various inflammatory conditions. So far, in vitro leukocyte aggregation has been studied by mixing patients' plasma or serum with leukocytes of a normal donor in a platelet aggregometer. We evaluated the leukergy phenomenon, that is, the occurrence of aggregated leukocytes in peripheral blood of patients with inflammatory disorders, as an alternative tool to the former method. Leukocyte aggregation could be induced by zymosan-activated plasma, phorbol myristate acetate, and formyl-methionyl-leucyl-phenylalanine, as well as by the calcium ionophore A23187 in vitro in whole human blood. This aggregation was blocked by various lipoxygenase and cyclooxygenase pathway inhibitors. Leukergy was diagnosed in peripheral blood of patients with inflammatory disorders and was generally not associated with elevated concentrations of immune complexes, lactoferrin, C3a des Arg, or C5a des Arg. To the best of our knowledge, this is the first report on leukocyte aggregation studies performed in whole blood. Such study permits evaluation of the aggregation phenomena of leukocytes in their natural milieu. Furthermore, it is possible with this method to obtain direct visualization of leukocytic aggregates in the peripheral blood of patients. The significance of our results and their possible implications are discussed in light of the pertinent literature.
Adult↗
Polymorphonuclear leukocyte chemotaxis in systemic lupus erythematosus.
Studies completed thus far indicate that normal human serum contains a specific anionic polypeptide (cochemotaxin) that permits low concentrations of C5a desarg to exhibit chemotactic activity for human polymorphonuclear leukocytes. Under conditions of limited complement activation in vivo, the cochemotaxin may play an important physiologic role by amplifying the chemotactic activity of C5a desarg. The "complex" of C5a desarg plus cochemotaxin probably accounts for most of the chemotactic activity that is generated in human serum after complement activation and also appears to be the target of the heat stable cationic protein inhibitor (Bb fragment of Factor B) that is present in serum from some patients with active systemic lupus erythematosus.
Chemotaxis, Leukocyte↗
Chemotactic responses of human peripheral blood monocytes to the complement-derived peptides C5a and C5a des Arg.
We examined responses of human peripheral blood polymorphonuclear leukocytes (PMN) and monocytes to the highly purified human complement-derived peptides C5a and C5a des Arg. As reported previously, C5a proved to be approximately 10- to 20-fold more potent than C5a des Arg as a chemoattractant for human PMN. C5a also was more potent than C5a des Arg in causing PMN to acquire a polarized morphology. In contrast, we found that human monocytes do not distinguish between C5a and C5a des Arg when these peptides are used as chemoattractants. In two different assay systems, both peptides acted at identical concentrations to stimulate suboptimal and optimal migration of monocytes. Human monocytes also did not distinguish between C5a and C5a des Arg when these peptides were used as inducers of polarization. Studies performed with functionally active, [125I]-labeled C5a and C5a des Arg, however, demonstrated that binding of C5a des Arg to monocytes differed from binding of C5a. Although [125I]-C5a des Arg appeared to bind to the same receptor as [125I]-C5a, binding of labeled C5a des Arg occurred with an affinity that was approximately 100-fold less than that observed with labeled C5a. These results indicate that leukocyte chemotactic and polarization responses to C5a and C5a des Arg vary, depending on the target cell type.
Adult↗
Evidence for a role of the amino-terminal region in the biological activity of the classical anaphylatoxin, porcine C5a des-Arg-74.
The presence of methionyl residues at positions 1 and 17 in porcine classical anaphylatoxin (e.g. C5a des-Arg-74) permits chemical cleavage of the factor with cyanogen bromide to generate two defined fragments. Peptides corresponding to the amino-terminal fragment, CN-I, and the carboxyl-terminal peptide, CN-II, were purified from the CNBr digest of C5a des-Arg-74 by reverse-phase high performance liquid chromatography. The isolated derivatives were assessed for their abilities to cause contraction of isolated guinea pig ileal smooth muscle, guinea pig lung parenchymal strips, and degranulation of guinea pig polymorphonuclear neutrophils. In each assay, CN-I was devoid of biological activity at concentrations greater than 10(-6) M. In contrast, the carboxyl-terminal 56-residue fragment, CN-II, possessed weak (10(-6) versus 10(-9) M for classical anaphylatoxin) agonist activity in each of the assay systems. Our data suggest that structural information contained in the amino-terminal 17 residues of the C5a des-Arg-74 molecule contributes to the biological potency of the intact factor, but is not an essential component of the active site. Whether the structural information in residues 1-17 relates to receptor binding directly or serves to stabilize the conformation of the 18-73-fragment containing the active center of the molecule is yet to be determined.
Amino Acids↗
Loss and recovery of sensitivity of guinea-pig isolated ileum to the spasmogenic action of the complement peptide C5adesArg.
Deactivation (tachyphylaxis) of the guinea-pig isolated ileum to the spasmogenic action of the complement peptide C5adesArg was analysed. It appeared to consist of 2 components: a fast one, characterized by rapid onset of deactivation and by recovery within 2-3 min (see Damerau et al., 1985b), and a slow component, characterized by progressively increasing loss of sensitivity (until complete deactivation after several minutes) and by recovery within about 80 min. Slow deactivation shows an exponential time course; it is dependent on concentration as well as contact time with C5adesArg and occurs under conditions (incubation in Ca2+-free medium or at 16 degrees C) in which the peptide has no spasmogenic effect. Recovery from slow deactivation follows an exponential time course at 34 degrees C but is blocked at 16 degrees C; on average it reaches about half of the initial sensitivity. The results indicate that the slow deactivation is mainly due to blockade of C5a receptors by the ligand and is independent of the spasmogenic effect of C5adesArg.
Animals↗
Pharmacological characterization of the slow component of deactivation of guinea-pig isolated ileum to the spasmogenic action of C5adesArg.
The slow component of deactivation of guinea-pig isolated ileum to C5adesArg was studied to analyse the mechanism of loss and subsequent recovery of sensitivity. Neither cycloheximide (10(-3) M) nor colchicine (5 X 10(-5) M), vinblastine, lumicolchicine, or cytochalasin B (each 2 X 10(-5) M) affected significantly the spasmogenic effect of C5adesArg or the course of deactivation produced by repeated applications; chloroquine (2 X 10(-4) M) inhibited the spasmogenic effect unspecifically without interfering with deactivation. Recovery from slow deactivation was totally blocked by chloroquine and considerably diminished by colchicine and vinblastine, but was not affected by the other agents. It is proposed that recovery involves lysosomal processing of C5a receptors (occupied by the peptide) but does not require biosynthesis of new receptors.
Animals↗
Complement activation and clearance in acute illness and injury: evidence for C5a as a cell-directed mediator of the adult respiratory distress syndrome in man.
The appearance of the adult respiratory distress syndrome (ARDS) during the course of acute illness is believed to result, in part, from intrapulmonary neutrophil sequestration and degranulation induced by circulating inflammatory mediators. To evaluate the role of complement-neutrophil interactions in the pathogenesis of ARDS in man, 34 patients suffering from intra-abdominal sepsis (seven), multisystem trauma (15), or acute pancreatitis (12) were serially studied with regard to neutrophil migratory responses to C5a and F-Met-Leu-Phe, lysosomal content of beta-glucuronidase and lysozyme, and simultaneously obtained plasma levels of immunoreactive C3adesArg and C5adesArg. Nineteen patients developed ARDS. In these patients, plasma C3adesArg levels obtained within 72 hours of admission to the hospital were elevated to 305 +/- 35 ng/ml compared with 145 +/- 16 ng/ml for patients who did not develop ARDS (p less than 0.0005). C5adesArg levels were not elevated in either group. In vitro studies showed that neutrophils from normal persons were able to clear all of the C5a/C5adesArg generated in up to 5% zymosan-activated serum, while no clearance of C3adesArg was identified. Patient migratory responses could be divided into three groups based on their initial (less than 72 hour) samples: (1) hyperresponsive to both N = formyl-methionyl-leucyl-phenylalanine (FMLP) and C5a, (2) specifically deactivated to C5a, and (3) deactivated to both C5a and FMLP. Patients in the latter two groups developed ARDS. Enzyme content of neutrophils from patients who developed ARDS showed a substantial fall in beta-glucuronidase and lysozyme levels. The finding of elevated plasma C3a levels and deactivation of migratory response to C5a support the contention that complement activation had occurred in these patients and that their neutrophils had been exposed to C5a/C5adesArg in vivo. The finding of nonspecific migratory dysfunction associated with lysozymal enzyme loss, a circumstance not reproducible in vitro by C5a exposure, suggests that other stimuli produced degranulation of neutrophils made hyperresponsive by prior exposure to C5a.
Chemotaxis, Leukocyte↗
Various authentic chemoattractants mediating leukocyte adherence inhibition.
This study resulted from the finding that cancer extract-induced leukocyte adherence inhibition (LAI) depends on release of arachidonic acid metabolites of which leukotriene B4 (LTB4) is a chemoattractant. Leukotrienes, the chemotactic fragment of the fifth component of complement (C5a des arg), N-formyl-L-methionylyl-L-leucyl-L-phenylalanine (FMLP), platelet-activating factor (PAF), and phorbol myristate acetate (PMA) increased the nonadherence of human leukocytes to glass with bell-shaped dose-response curves. For maximum nonadherence, the optimum concentration was about 2 X 10(-11) M LTB4, 10(-9) M C5a des arg, 2 X 10(-8) M FMLP, 2 X 10(-9) M PAF, and 2 X 10(-8) M PMA. Of the adherent leukocytes, 29% became nonadherent. Higher concentrations of leukotriene E4, FMLP, PAF, and PMA induced leukocyte hyperadhesiveness. Chemoattractant-induced LAI was antagonized by inhibitors of glycolysis, oxidative metabolism, electron transport, microfilaments, and microtubules, whereas the same concentration of inhibitors did not alter the adherence of leukocytes to glass in control tubes. T-cells, neutrophils, and mononuclear cells showed LAI to LTB4, C5a des arg, and FMLP. Moreover, no additive effect was observed when leukocytes exposed to a first chemoattractant at its optimum concentration were exposed to a second. A competitive inhibitor of leukotriene slow-reacting substance of anaphylaxis, FPL 55712, blocked in a dose-response fashion LTB4-, C5a des arg-, and FMLP-triggered LAI. Eicosatetraenoic acid and nordihydroguaiaretic acid, cyclo-oxygenase-lipoxygenase pathway inhibitors, also markedly antagonized LTB4-, C5a des arg-, and FMLP-triggered LAI. Indomethacin (10(-5) M), piroxicam (10(-6) M), and aspirin, cyclooxygenase antagonists, negated LTB4-, C5a des arg-, and FMLP-induced LAI. Prostaglandins E2, E1, and F2 and prostacyclin did not trigger LAI. However, selective thromboxane synthetase antagonists completely negated chemoattractant-triggered LAI. Our studies indicated that chemoattractants stimulated leukocytes to produce thromboxanes, which increased leukocyte nonadherence.
Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
The in vivo effect of leukotriene B4 on polymorphonuclear leukocytes and the microcirculation. Comparison with activated complement (C5a des Arg) and enhancement by prostaglandin E2.
The effect of synthetic leukotriene B4 (LTB4) on chemotaxis in vivo (51Cr-polymorphonuclear leukocyte [PMN] accumulation) was examined and its potency compared with that of C5a des Arg-containing zymosan-activated plasma (ZAP). On a molar basis the amount of C5a des Arg calculated to be in our preparation of ZAP was found to be up to approximately 80 times more potent than LTB4, although in vitro the two chemotaxins have been reported to be about equipotent. ZAP is more representative of what may happen in vivo than its principal constituent C5a des Arg, but for a more precise comparison the purified and isolated peptide will have to be compared with synthetic LTB4. Whereas ZAP induced severe PMN-dependent microvascular injury (increase in vessel permeability [125I-albumin] and hemorrhage [59Fe-erythrocytes]), LTB4 only induced an increase in vascular permeability, and this occurred only in the presence of simultaneously injected prostaglandin E2 (PGE2). PGE2 also enhanced substantially the number of PMNs and the amount of exuded plasma at injection sites of the chemotaxins. However, unlike in two other reports, LTB4 did not cause an immediate transient increase in vessel permeability, nor did it enhance the permeability-increasing effect of bradykinin. Furthermore, unlike PGE2 LTB4 did not induce an increase in blood flow, but a decrease (57Co-microspheres). It is concluded that LTB4 may act as a host-derived chemoattractant in vivo, but, compared with that of ZAP (primarily activated complement), its role in acute inflammation is probably less significant than that of the complement-derived chemotaxin(s).
Animals↗
Effect of dialyzer reuse on complement activation and neutropenia in hemodialysis.
A prospective study of 10 patients undergoing hemodialysis showed that less neutropenia and complement activation occurred with dialyzer reuse. Neutrophil counts fell 95% +/- 5% (SEM) with first use and 66% +/- 8% and 48% +/- 10% with second and third uses, respectively (p less than 0.05). The production of complement component C5a-desarg, as measured by the bioassay granulocyte aggregation, was decreased by 96% +/- 1% and 93% +/- 2% with second and third uses, respectively (p less than 0.05). We investigated the role of the dialyzer disinfectant formaldehyde in decreased neutropenia. In vitro, formaldehyde inhibited granulocyte aggregation and chemotaxis and the dialyzer membrane's ability to generate granulocyte aggregating activity; however, this occurred only at concentrations higher than those likely to obtain in patients. The ability of dialysis membranes to generate granulocyte aggregating activity in plasma was decreased 55% +/- 5% by their prior sequential preincubation in plasma and then formalin (p less than 0.05) and extensive rinsing, which is similar to the circumstances obtaining with dialyzer reuse. Preincubation of membranes in plasma or formalin alone resulted in no change in the membrane's ability to generate granulocyte aggregating activity. We conclude that the exposure of membranes to both plasma and formalin during dialysis and storage is responsible for the decreased C5a-desarg production with reuse, probably because plasma proteins are fixed to the membrane in such a way that interrupts free interaction between the membrane and plasma complement components.
Agranulocytosis↗
Complement activation and stimulation of chemotaxis by Chlamydia trachomatis.
The stimulus for the migration of polymorphonuclear leukocytes (PMNs) in acute chlamydial infection was studied in vitro by examining the chemotaxigenic effect of L2 and DE Chlamydia trachomatis elementary bodies (EB) upon the plasma of three healthy donors. In each individual experiment, chemotactic response was assessed with PMNs and plasma from the same respective donor, and no specific antibodies against C. trachomatis were detected in the plasma of any donor. Chemotaxis was observed in an agarose plate assay and was quantitated as the chemotactic differential, or CD (directed migration of PMNs minus random movement of PMNs). For each donor, the mean CD was significantly greater (P less than 0.005) when plasma preincubated for 2 h with L2 EB was used as the chemoattractant than when (i) plasma alone, (ii) plasma preheated to 56 degrees C for 30 min before incubation with L2 EB, or (iii) L2 EB in phosphate-buffered saline (PBS) was used as the potential chemoattractant. Similarly, in the one donor in whom DE EB were studied, the mean CD was also significantly greater (P less than 0.005) for plasma preincubated with DE EB as compared with (i) plasma alone or (ii) DE EB in PBS. Complement activation by C. trachomatis EB was assessed by radioimmunoassay for C5a des-arginine in all chemoattractant preparations used in the chemotaxis assay. Mean C5a des-arginine levels were high in plasma samples preincubated with L2 EB (171.00 +/- 10.64, 107.00 +/- 4.76, and 89.70 +/- 1.74 ng per ml) or DE EB (37.40 +/- 15.76 ng per ml) but were undetectable (less than 4.0 ng per ml) in (i) plasma alone, (ii) preheated plasma incubated with L2 EB, and (iii) PBS containing L2 EB. Thus, L2 EB and DE EB of C. trachomatis exert a chemotaxigenic effect upon normal antibody-negative plasma, and this effect is at least in part a result of complement activation and generation of the potent chemotaxin C5a.
Chemotaxis, Leukocyte↗